My protein has no protease (relevant to the E. coli system)degradation site, as I have checked on expasy. I store my protein at 4 degrees as it aggregated at -20 degrees. It is degrading over time and the band which was at 25 kDa earlier is getting thicker near the 18 kDa band. I have attached the SDS-PAGE picture.
(My protein is a fused protein in which I have fused the protein with an antimicrobial peptide)
Not all proteases have defined sites, so you can certainly still have protease degradation without the site. There can also be spontaneous cleavage at exposed regions of some proteins. So I would worry less about the why and find a workaround. Adding protease inhibitors might help, or storing at -80 might help.
Dear Jyoti Rustagi
just some questions to better undestanrd the possible issue:
is you protein an enzime (eg contain protease domain) that can have autocleavage activity?
Is it contain cysteines that are theoretically involved in S-S bond formation?
Did you tried to ad EDTA after elution or other protease inhibitors?
Did you tried to add glycerol (20% is enough) to prevent aggregation of your protein when is it stored at -20°C?
Can you share with us some more element of the cell lysis and purification protocoll? Is the degradation already present at the end of the purification as seems from your SDS-page?
best regards
Manuele
Manuele Martinelli
My protein is a lysozyme-like protein.
It only contains a single cysteine residue.
I have not tried EDTA as I am using NI-NTA for purification.
No, I have not used 20% glycerol, I started storing it at 4.
Cell lysis: 20mM HEPES, 200mM NaCl, 1mM MgCl2, 2% TX100, 5% Glycerol.
1.5g pellet (12 hours grown culture at 18 degrees).
Sonication at 45 Amplitude for 4 min(5 sec on & 7 sec off) on ice.
after lysis when run crude cell lysate on SDS PAGE, there is no degradation but it is seen after purification.
Dear Jyoti Rustagi
if at the moment of elution the protien is not degraded but degradation happen later during the 4°C incubation you can try to:
- Add EDTA 5-10mM after IMAC elution
or
- Add 20% of glycerol, aliquot and froze it at -20°C
good luck
Manuele
I used Pierce Protease inhibitor cocktail in crude lysate, it helped in stopping the degradation to an extent.
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